Improvement of nested reverse transcription-polymerase chain reaction (RT-PCR) with high specificity and sensitivity detection of sapovirus in food matrix

Sapovirus (SaV) is a causative agent of human gastroenteritis in both community outbreaks and sporadic cases worldwide. Shellfish accumulate a variety of pathogens during filter feeding. In particular, the contamination of shellfish by SaV has caused several outbreaks. As reported previously, nested RT-PCR (nRT-PCR) has been widely used in clinical samples, but has not proven suitable for food samples, such as oysters. This study aimed to identify a primer set for the detection of SaV with high specificity and sensitivity in food samples. To accomplish this, primers were improved in RNA-dependent RNA polymerase (RdRp) regions of SaV whole genome sequences. The sensitivity of the improved nRT-PCR was 100–1000 times higher than that of previous nRT-PCR and > 10 times higher than that of the previous real-time RT-PCR assay. Notably, cross-reaction with other viruses or food matrices was not observed by the specificity test. This study improved the reliable primer set to detect SaV in various food matrices with high sensitivity. •This study developed the new primer set to detect sapovirus in oysters.•The primer showed more than 100-fold improved sensitivity.•The primer had no cross-reactivity with food and other viruses.•The improved primer can reliably detect food-contaminated sapovirus.